In 1975 Kohler and Milstein reported the generation of the first monoclonal antibody. Their landmark paper describes the methods for fusing antibody-producing B cells, isolated from the spleens of immunized mice, with aggressively proliferating mouse myeloma cells. This resultant hybrid cell, a so-called hybridoma, possesses the characteristics of both parental cells; it produces and secretes large amounts of antibody during its continued growth and proliferation. Through a series of systematic cellular dilutions, genetically singular hybridoma cells are isolated that produce an antibody of singular isotype specificity, a so-called monoclonal antibody (mAb).
Due to their exquisite specificity, mAbs held the promise for developing “magic bullet” therapies for treating human disease. Nevertheless, over the past 40 years a mere handful of human mAbs have been developed into therapeutics. To understand the reason for this apparent failure, one must appreciate the events occurring during the in vivo antibody response, and how past attempts to replicate them for developing human mAbs were unsuccessful.
The most common procedure for generating monoclonal antibodies starts with the immunization of an animal with the antigen of interest. The antigen, draining into a local lymph node or spleen, activates naïve B cells to proliferate and produce IgM antibodies against the antigen. These activated B cells are then instructed by antigen-activated CD4+ T-cells to undergo a process known as class switching. During class switching, the B cell immunoglobulin gene is reorganized, resulting in a switch in the type of antibody produced from low-affinity IgMs to high affinity IgGs.
As the antibody response progresses, the progeny of the original parental B cells continue to proliferate in the lymph node and spleen to give rise to a structure known as the germinal center. Within the germinal center, proliferating B cells are exposed to additional cellular and chemical signals that induce the Bcells to undergo somatic hypermutation and affinity maturation. During somatic hypermutation, point mutations are introduced into the immunoglobulin variable region gene sequences that alter the antibody's affinity for binding the antigen. During affinity maturation, B cells expressing antibodies with higher antigen affinities continue to proliferate and are signaled to differentiate into either plasma cells or memory B cells. B cells possessing deleterious mutations are deleted by apoptosis. Typically, at this stage of mAb development, B cells are isolated from the lymph node or spleen of the immunized animal, and are fused with species-specific myeloma cells. The fused cells are allowed to grow to produce antigen specific IgG antibodies, which are screened for potential use in human therapeutics.
The early success of this technology in animals prompted scientists in the 1980's to extend this concept for the production of human mAbs. However, extrapolation from animal to man was fraught with difficulties. The first hurdle investigators faced was the lack of antigen specific B cells. Recall that under standard procedures, antigen specific B cells are typically harvested from immunized animals; a method not generally applicable to humans unless the long-term safety of the antigen used for immunization is known. This problem is further compounded by (i) the fact that there is no ready source of activated B cells, and (ii) the inability to obtain either lymph nodes or spleens from human subjects. These factors prompted the development of a variety in vitro strategies to produce human monoclonal antibodies.
Although initial results showed great promise, the inability of past technologies to completely reconstruct the sequence of events of the in vivo antibody response ultimately caused them to fail. To date three technologies have been developed to address these challenges and are currently used for the development of human monoclonal antibody therapeutics.
The oldest of these technologies is the humanization of murine monoclonal antibodies to form a human mouse chimeric (i.e., humanized) antibody. Using this technology, murine monoclonal antibodies to a putative human antigen are generated in the traditional methods of Kohler and Milstein. Nevertheless, such antibodies have little to no utility as human therapeutics since they are generated in mice and thus would elicit a human anti mouse antibody response (HAMA response) in humans. To reduce the immunogenicity of the murine monoclonal antibody the FAb (fraction antigen binding) fragment of the murine mAb was chemically weaved into the structure of a human antibody molecule. Although these humanized antibodies were less immunogenic in people, the murine segments still posed a challenge due to their residual immunogenicity.
A second technology, Phage Display, uses vast phage libraries expressing random sequences of the human antibody variable region. These libraries are screened to select specific vectors that will bind a putative human antigen target. Once identified the specific bacteriophage are grown and then processed to collect the FAb domain. Although this technology generates antibodies that are fully human, the process requires library screening and multiple cloning steps to achieve a fully human antibody.
The transgenic mouse represents the final technology that is currently used to generate fully human antibodies. Simply put, these mice have been genetically engineered to contain the fully human equivalent of the genes that control the murine immune response. This technology seems to address all of the prior pitfalls of human mAb development since the putative therapeutic is of fully human origins and thus should not elicit an antibody response. Nevertheless, although the transgenic mouse technology has existed since 1993, fully human monoclonal antibodies generated are not commonplace.
What is needed therefore, are effective compositions and methods for the generation of antibodies. More specifically, what is needed are compositions and methods for the generation of species specific antibodies (for example, fully human antibodies against human target antigens). Such methods should comprise the efficient and effective presentation of antigens to the appropriate components of the immune systems. Preferably, such methods should be species specific, promoting for example, the generation of human antibodies for use in humans without eliciting undesired immunogenic reactions. What is also needed are methodologies that do not cause unwanted side effects in the entire organism. In addition what is needed are methods for generating human anti-human monoclonal antibodies from peripheral human blood lymphocytes wherein such antibodies not only bind the human antigen, but also have been shown to neutralize the biologic action of the putative antigen.